Photolithography and direct photochemical unblocking are techniques used in microarray technology to build up arrays of oligonucleotides or peptides binding agents at defined locations on solid support. Although this approach is employed to generate arrays for use in some analytical applications based on DNA-hybridisation, the high cost of photolithographic masks, the use of non-commercial photolabile phosphoramidite monomers and problems with quantitative, direct photochemical unblocking lead to very expensive and often poor quality microarrays30.
One attempt to solve these problems is based on so-called chemical amplification. This technique is borrowed from the electronics industry, and uses a combination of photochemically and thermally generated acid production at desired sites on an array surface covered with a diffusion-limiting polymeric film31. However, the need to use strong acids such as benzenesulphonic acid in the key photodirected step, while acceptable in the production of intergrated circuits, makes application of this approach with acid labile purine nucleosides difficult32,33. There have been attempts to overcome these problems by designing new photoacid generating compounds, but on the whole, these attempts have not been successful. By way of example, Le Proust et al28 and Gao et al27 have employed photolabile hexafluoroantimonates in the synthesis of oligonucleotides in solution (see also WO99/41007). However, these photoacid generators involve the release of free radicals which can result in undesirable side reactions such as blockage of 5′-OH group.
Reichmannis et al34 described 2-nitrobenzyl esters which photolyse to produce trimethylacetic acid, and more particularly the mechanism of this reaction and its yield, both in solution and in a polymer matrix. WO00/66259 describes the use of photoactivated reagents which when activated are capable of removing protecting groups at the termini of substrates being synthesised on a solid phase. The application suggests the use of triarylsulphonium hexafluorantimonates, triarylsulphonium hexafluorophosphates, 2,1,4-diazonapthoquinone sulphonates and perhalogenated traiazines. In one example, 1-[2-nitrophenyl]ethyl-1-trichloroacetate is irradiated with UV light to generate trichloroacetic acid for removing protecting 5′-dimethoxytrityl groups from oligonucleotide substrates.
The actual fabrication of arrays of binding agents and in particular oligonucleotide or peptide arrays is an area of intense interest in the art. The chemical synthesis of binding agents such as oligonucleotides is well known. The most commonly used method is a solid phase synthesis using controlled porosity glass or equivalent material as the support. Stepwise extension of an oligonucleotide attached via a linker molecule to the support occurs by addition of one nucleotide at a time. Attachment to the linker is commonly through the oligonucleotide-3′-OH, with chain extension therefore at the oligonucleotide-5′-OH. Because of the stepwise nature of the process, satisfactory synthesis of oligonucleotides of commonly desired chain lengths of 20 or more nucleotides requires a high stepwise yield. The overall yield of an N-mer synthesised with a stepwise yield of Y is YN, and diminishes rapidly once Y falls beneath about 0.95. After completion of synthesis the oligonucleotide is cleaved from the support and purified prior to use. Caruthers35 and Beaucage & Iyer36 have written detailed reviews of oligonucleotide synthesis methods.
Typically, each new oligonucleotide monomer is added to a growing oligonucleotide as a modified nucleotide substituted at its 5′-OH position with a 4,4′-dimethoxytrityl group, and a beta-cyanoethyl-phosphoramidite at its 3′-OH position. Synthesis starts by coupling the first nucleotide through its 3′-OH group to the terminal hydroxyl group of a linker molecule attached to a solid support. Any unreacted terminal OH groups are then blocked with acetic anhydride, and the trivalent phosphite is oxidised to pentavalent phosphate. The dimethoxytrityl group is then removed with acid from the 5′-OH position of the first nucleotide, which can then react with the next nucleotide phosphoramidite to be added. The cycle of steps is then repeated until the desired chain length has been synthesised. Finally, alkali treatment is used to remove N-protective groups and also to cleave an alkali-labile bond in the linker, thereby releasing the oligonucleotide which may then be purified.
In prior art solid phase synthesis methods, removal of DMT, or detritylation, is effected with di- or trichloroacetic acid according to equation (1). Stronger acids cause chain breakage by depurination. It should be noted that protons are reagents, not catalysts, in the detritylation reaction, and are consumed with a stoichiometry of 1 proton per DMT+ cation released, as in equation (1):oligonucleotide-5′-O-DMT+H+=oligognucleotide-5′-OH+DMT+  (1)
The repetitive cycle of steps in the synthesis of oligonucleotide, or indeed peptide, lends itself to automation, and a variety of commercially available instruments have been developed for that purpose. The availability of synthetic oligonucleotides has led to the development of arrays of oligonucleotides on paper or other polymeric sheets, fabric or glass, allowing multiple hybridisation reactions to be carried out in parallel.
Early descriptions of oligonucleotide arrays were from academic laboratories and the array densities achieved were modest. Construction methods define three classes of array, namely (a) arrays printed from pre-synthesised oligonucleotides, (b) arrays synthesised in situ by reagent printing and (c) arrays synthesised in situ by a photodirected method.
The printing methods create array elements at a modest density, up to 5,000/cm2, with each element having a diameter of about 0.1 mm and separated from its neighbours by a similar distance and possibly an additional physical barrier to prevent reagents that should be constrained to selected elements from spreading to adjacent elements. Photolithographic methods currently achieve much higher densities (160,000/cm2), with the potential for even higher (106/cm2).
Photodirected synthesis of oligonucleotides in arrays was first described in 1991 by Fodor et al37. The main technical innovation was to replace the conventional acid-removable dimethoxytrityl blocking group at the oligonucleotide 5′-OH with a group that was photo-removable. The array elements at which groups would be unprotected, and therefore reactive with whichever A, C, G or T-deoxyribonucleotide-3′-O-phosphoramidite was subsequently applied, were determined by patterned illumination of the array surface. Proximity or contact photolithography is needed to minimise stray light, and requires numerous high precision physical masks (metal on glass or quartz), with the result that this technology has the significant disadvantages of high cost and low flexibility.
In the photodirected method for making arrays, the synthesis consists of a cycle of steps that adds a nucleotide at each chain length to photoselected array elements. The cycle is used four times (once each for A, C, G & T) to extend the length of the array by one nucleotide, and 4N times to make an array of N-mers. The first four cycles couple monomer to a linker attached to the glass or other solid surface. The linker has an aliphatic —OH group at its free end. All subsequent cycles couple monomer to oligonucleotide 5′-OH. The sequence of actions in the photodirected synthesis of an oligonucleotide array, using nucleotide monomers with photolabile protection of the 5′-OH group is given in Table 1.
The use of contact or close proximity photolithography uses masks of metal on glass or quartz. The transmission of light through the metallised areas of the mask is 10−5 of that transmitted through the clear (non-metallised) areas (Pease et al29). In other words, the contrast ratio of metal on glass masks is 105. The associated intensity level of stray light is negligible in the context of photodirected synthesis of oligonucleotide arrays. However, the masks are expensive, and the number needed is large (100 for a 25-mer array), making this method of fabricating arrays unsuitable for use outside an expensively equipped industrial environment.
To overcome this drawback of expense and inflexibility, several groups (Singh-Gasson et al38, Garner39 and Staehler40) have reported the use of projection photolithography using Digital Micromirror Device (DMD: Hornbeck41) projectors) in association with photosensitive blocking groups of the oligonucleotide-5′-OH group. The aim was to avoid the cost and inflexibility of metal-on-glass physical masks by replacing them with programmable masks in silico that determine the patterned output of a light projector. LeProust et al28 have also used a DMD projector, but used photoacid generation to deprotect the tritylated oligonucleotide-5′-OH group.
Furthermore, despite the work described above, it remains a problem in the art in generating acid for in situ deprotection of oligonucleotides.